Modeling Multi-Step Organic Reactions: Can Density Functional Theory Deliver Misleading Chemistry?

Many organic reactions are characterized by a complex mechanism with a variety of transition states and intermediates of different chemical natures. Their correct and accurate theoretical characterization critically depends on the accuracy of the computational method used. In this work, we study a complex ambimodal cycloaddition with five transition states, two intermediates, and three products, and we ask whether density functional theory (DFT) can provide a correct description of this type of complex and multifaceted reaction. Our work fills a gap in that most systematic benchmarks of DFT for chemical reactions have considered much simpler reactions. Our results show that many density functionals not only lead to seriously large errors but also differ from one another in predicting whether the reaction is ambimodal. Only a few of the available functionals provide a balanced description of the complex and multifaceted reactions. The parameters varied in the tested functionals are the ingredients, the treatment of medium-range and nonlocal correlation energy, and the inclusion of Hartree-Fock exchange. These results show a clear need for more benchmarks on the mechanisms of large molecules in complex reactions.

The interaction of thiocyanate with peptides-A computational study.

According to the Hofmeister series, thiocyanate is the strongest "salting in" anion. In fact, it has a strong denaturant activity against the native state of globular proteins. A molecular level rationalization of the Hofmeister series is still missing, and therefore the denaturant activity of thiocyanate also awaits a robust explanation. In the last years, different types of experimental studies have shown that thiocyanate is capable to directly interact with both polar and nonpolar groups of polypeptide chains. This finding has been scrutinized via a careful computational procedure based on density functional theory approaches. The results indicate that thiocyanate is able to make H-bonds via both the nitrogen and sulfur atom, and to make strong van der Waals interactions with almost all the groups of polypeptide chains, regardless of their polarity.

Disentangling the Puzzling Regiochemistry of Thiol Addition to o-Quinones.

The addition of thiol compounds to o-quinones, as exemplified by the biologically relevant conjugation of cysteine to dopaquinone, displays an anomalous 1,6-type regiochemistry compared to the usual 1,4-nucleophilic addition, for example, by amines, which has so far eluded intensive investigations. By means of an integrated experimental and computational approach, herein, we provide evidence that the addition of glutathione, cysteine, or benzenethiol to 4-methyl-o-benzoquinone, modeling dopaquinone, proceeds by a free radical chain mechanism triggered by the addition of thiyl radicals to the o-quinone. In support of this conclusion, DFT calculations consistently predicted the correct regiochemistry only for the proposed thiyl radical-quinone addition pathway. These results would prompt a revision of the commonly accepted mechanisms for thiol-o-quinone conjugation and stimulate further work aimed at assessing the impact of the free radical processes in biologically relevant thiol-quinone interactions.

Proton-Sensitive Free-Radical Dimer Evolution Is a Critical Control Point for the Synthesis of Δ2,2'-Bibenzothiazines.

The mechanism of the acid-dependent interring dehydrogenation in the conversion of the single-bonded 3-phenyl-2H-1,4-benzothiazine dimer 2 to the Δ2,2'-bi(2H-1,4-benzothiazine) scaffold of red hair pigments is disclosed herein. Integrated chemical oxidation and oxygen consumption experiments, coupled with electron paramagnetic resonance (EPR) analyses and DFT calculations, allowed the identification of a key diprotonated free-radical intermediate, which was implicated in a remarkable oxygen-dependent chain process via peroxyl radical formation and evolution to give the Δ2,2'-bi(2H-1,4-benzothiazine) dimer 3 by interring dehydrogenation. The critical requirement for strongly acidic conditions was rationalized for the first time by the differential evolution channels of isomeric peroxyl radical intermediates at the 2- versus 3-positions. These results offer for the first time a rationale to expand the synthetic scope of the double interring dehydrogenation pathway for the preparation of novel symmetric double-bond bridged captodative heterocycles.

An ab initio study of Cu-based delafossites as an alternative to nickel oxide in photocathodes: effects of Mg-doping and surface electronic features.

CuMO2 delafossites (M = Al, Ga, and Cr) are p-type semiconductor oxides that have been recently proposed as the electrode in p-type dye-sensitized solar cells (p-DSSC) which is an alternative to the standard, low-performing nickel oxide. To assess this potential application of delafossites, we report here a DFT-based investigation of the structural and electronic properties of CuAlO2, CuGaO2 and CuCrO2. In particular, we address the role of Mg doping to obtain the p-type semiconducting character: the substitution of an M3+ cation with Mg2+ is easier with Ga than with Al and Cr, and, in all cases, the hole introduced by Mg2+ leads to the formation of Cu2+ species. Moreover, we address surface electronic features in order to characterize the most exposed delafossite surface termination and, more importantly, to predict the valence band maximum energy value, which determines the p-DSSC open circuit potential. From analysis of all our results, CuGaO2 emerges as the most promising system that can boost the development of new photocathodes for p-DSSCs.

Hot spot mapping of protein surfaces with TEMPOL: Bovine pancreatic RNase A as a model system.

TEMPOL spin-label has been used to identify surface exposure of protein nuclei from NMR analysis of the induced paramagnetic relaxation enhancements (PRE). The absence of linear dependence between atom depths and observed PRE reveals that specific mechanisms drive the approach of the paramagnet to the protein surface. RNase A represents a unique protein system to explore the fine details of the information offered by TEMPOL induced PRE, due to the abundance of previous results, obtained in solution and in the crystal, dealing with surface dynamics behavior of this protein. MD simulations in explicit solvent have been performed, also in the presence of TEMPOL, in order to delineate the role of intermolecular hydrogen bonds (HB) on PRE extents. Comparison of our results with the ones obtained from multiple solvent crystal structure (MSCS) studies yields information on the specificities that these two techniques have for characterizing protein-ligand interactions, a fundamental step in the development of reliable surface druggability predictors.

Antimicrobial potency of cationic antimicrobial peptides can be predicted from their amino acid composition: Application to the detection of "cryptic" antimicrobial peptides.

Cationic antimicrobial peptides (CAMPs) are essential components of innate immunity. Here we show that antimicrobial potency of CAMPs is linearly correlated to the product CmHnL where C is the net charge of the peptide, H is a measure of its hydrophobicity and L its length. Exponents m and n define the relative contribution of charge and hydrophobicity to the antimicrobial potency. Very interestingly the values of m and n are strain specific. The ratio n/(m+n) can vary between ca. 0.5 and 1, thus indicating that some strains are sensitive to highly charged peptides, whereas others are particularly susceptible to more hydrophobic peptides. The slope of the regression line describing the correlation "antimicrobial potency"/"CmHnL product" changes from strain to strain indicating that some strains acquired a higher resistance to CAMPs than others. Our analysis provides also an effective computational strategy to identify CAMPs included inside the structure of larger proteins or precursors, which can be defined as "cryptic" CAMPs. We demonstrate that it is not only possible to identify and locate with very good precision the position of cryptic peptides, but also to analyze the internal structure of long CAMPs, thus allowing to draw an accurate map of the molecular determinants of their antimicrobial activity. A spreadsheet, provided in the Supplementary material, allows performing the analysis of protein sequences. Our strategy is also well suited to analyze large pools of sequences, thus significantly improving the identification of new CAMPs and the study of innate immunity.

Replacing Nitrogen by Sulfur: From Structurally Disordered Eumelanins to Regioregular Thiomelanin Polymers.

The oxidative polymerization of 5,6-dihydroxybenzothiophene (DHBT), the sulfur analog of the key eumelanin building block 5,6-dihydroxyindole (DHI), was investigated to probe the role of nitrogen in eumelanin build-up and properties. Unlike DHI, which gives a typical black insoluble eumelanin polymer on oxidation, DHBT is converted to a grayish amorphous solid (referred to as thiomelanin) with visible absorption and electron paramagnetic resonance properties different from those of DHI melanin. Mass spectrometry experiments revealed gradational mixtures of oligomers up to the decamer level. Quite unexpectedly, nuclear magnetic resonance (NMR) analysis of the early oligomer fractions indicated linear, 4-, and 7-linked structures in marked contrast with DHI, which gives highly complex mixtures of partially degraded oligomers. Density functional theory (DFT) calculations supported the tendency of DHBT to couple via the 4- and 7-positions. These results uncover the role of nitrogen as a major determinant of the structural diversity generated by the polymerization of DHI, and point to replacement by sulfur as a viable entry to regioregular eumelanin-type materials for potential applications for surface functionalization by dip coating.

Stability of melamine-exfoliated graphene in aqueous media: quantum-mechanical insights at the nanoscale.

In recent experiments, melamine (1,3,5-triazine-2,4,6-triamine) has been proposed as an effective exfoliating agent to obtain high quality graphene from graphite. After washing out the melamine in excess, small amounts (ppm) are still needed to stabilize the dispersion of graphene flakes in aqueous media. To understand the origin of this behaviour, we investigated the melamine-graphene-water system and the fundamental interactions that determine its structure and energetics. To disentangle the subtle interplay of hydrogen-bonding and dispersive forces we used state-of-the-art ab initio calculations based on density functional theory. First, we focused on the case of water molecules interacting with melamine-graphene assemblies at different melamine coverages. We found that water-melamine interactions provide the driving force for washing off the melamine from graphene. Then, we addressed the interaction of single and double layers of water molecules with the graphene surface in the presence of an adsorbed melamine molecule. We found that this melamine acts as a non-covalent anchor for keeping a number of water molecules conveniently close to the graphene surface, thus helping its stabilization in aqueous media. Our analysis helps understanding how competing weak forces can lead to a stable graphene water suspension thanks to small amounts of adsorbed melamine. From our results, we derive simple indications on how the water-graphene interfacial properties can be tuned via non-covalent adsorption of small functional molecules with H-bond donor/acceptor groups. These new hints can be helpful to prepare stable graphene dispersions in water and so to unlock graphene potential in aqueous environments.

Towards eumelanin@zeolite hybrids: pore-size-controlled 5,6-dihydroxyindole polymerization.

5,6-Dihydroxyindole (1) and its N-methyl derivative (2), key eumelanin building blocks, were inserted into zeolite L by sublimation at 175 °C for 5 days. At a 10 mg/300 mg indole/zeolite ratio, the resulting hybrids displayed a stable deep red coloration. CP/MAS (13)C NMR and UV/Vis spectroscopy of the red species suggested the generation and accommodation of quinonoid biindole derivative(s) within the void space of the acidic zeolite channels. Removal of the zeolite matrix by treatment with HF gave a stable species that could be separated by HPLC and characterized by mass spectrometry as an oxygenated biindole derivative (or a mixture of isomers), suggesting addition of water to the original dimer and subsequent re-oxidation. The characterization was corroborated by optimized molecular geometries and simulated UV spectra with density functional calculations. Loading 1 or 2 into the larger pores of SBA-15 type mesoporous silica resulted in black eumelanin-type polymers, confirming channel size dependence over the polymerization process.

Free radical coupling of o-semiquinones uncovered.

As a rule, o-semiquinones decay through disproportionation leading to equimolar amounts of catechol and o-quinone products. However, the o-semiquinone 1S generated by pulse radiolysis oxidation of the eumelanin precursor 5,6-dihydroxyindole (1) decays with second-order kinetics to generate broad visible chromophores that are incompatible with the predicted absorption of 5,6-indolequinone (1Q). Using an integrated chemical, pulse radiolytic and computational approach as well as deuterium labeling, we show herein that 1S and related 5,6-dihydroxyindole semiquinones decay mainly by a free radical coupling mechanism. This conclusion was supported by the inverse kinetic isotope effect observed with deuterated 1S, the identification of unprecedented dihydrobiindole products by one-electron oxidation of 1, the good matching of simulated absorption profiles of free radical coupling products of 1S with experimental spectra, and a detailed computational analysis of the kinetics and thermodynamics of the disproportionation equilibrium and free radical coupling of 1S versus 1-1Q coupling. These results disclose, to the best of our knowledge, the first example of free radical dimerization of o-semiquinones outcompeting the classic disproportionation-driven catechol-quinone coupling and suggest that this hitherto unrecognized process may be of broader relevance than previously believed.

Computational 17O-NMR spectroscopy of organic acids and peracids: comparison of solvation models.

We examine several computational strategies for the prediction of the (17)O-NMR shielding constants for a selection of organic acids and peracids in aqueous solution. In particular, we consider water (the solvent and reference for the chemical shifts), hydrogen peroxide, acetic acid, lactic acid and peracetic acid. First of all, we demonstrate that the PBE0 density functional in combination with the 6-311+G(d,p) basis set provides an excellent compromise between computational cost and accuracy in the calculation of the shielding constants. Next, we move on to the problem of the solvent representation. Our results confirm the shortcomings of the Polarizable Continuum Model (PCM) in the description of systems susceptible to strong hydrogen bonding interactions, while at the same time they demonstrate its usefulness within a molecular-continuum approach, whereby PCM is applied to describe the solvation of the solute surrounded by some explicit solvent molecules. We examine different models of the solvation shells, sampling their configurations using both energy minimizations of finite clusters and molecular dynamics simulations of bulk systems. Hybrid molecular dynamics simulations, in which the solute is described at the PM6 semiempirical level and the solvent by the TIP3P model, prove to be a promising sampling method for medium-to-large sized systems. The roles of solvent shell size and structure are also briefly discussed.

Barrier Heights for Diels-Alder Transition States Leading to Pentacyclic Adducts: A Benchmark Study of Crowded, Strained Transition States of Large Molecules.

Theoretical characterization of reactions of complex molecules depends on providing consistent accuracy for the relative energies of intermediates and transition states. Here we employ the DLPNO-CCSD(T) method with core-valence correlation, large basis sets, and extrapolation to the CBS limit to provide benchmark values for Diels-Alder transition states leading to competitive strained pentacyclic adducts. We then used those benchmarks to test a diverse set of wave function and density functional methods for the absolute and relative barrier heights of these transition states. Our results show that only a few of the tested density functionals can predict the absolute barrier heights satisfactorily, although relative barrier heights are more accurate. The most accurate functionals tested are ωB97M-V, M11plus, ωB97X-V, PBE-D3(0), M11, and MN15 with MUDs from best estimates less than 3.0 kcal. These findings can guide selection of density functionals for future studies of crowded, strained transition states of large molecules.

Antioxidant Activities of Hydroxylated Naphthalenes: The Role of Aryloxyl Radicals.

Herein is delineated a first systematic framework for the definition of structure-antioxidant property relationships in the dihydroxynaphthalene (DHN) series. The results obtained by a combined experimental and theoretical approach revealed that 1,8-DHN is the best performing antioxidant platform, with its unique hydrogen-bonded peri-hydroxylation pattern contributing to a fast H atom transfer process. Moreover, the comparative analysis of the antioxidant properties of DHNs carried out by performing DPPH and FRAP assays and laser flash photolysis experiments, revealed the higher antioxidant power associated with an α-substitution pattern (i. e. in 1,8- and 1,6-DHN) with respect to DHNs exhibiting a ß-substitution pattern (i. e. in 2,6- and 2,7-DHN). DFT calculations and isolation and characterization of the main oligomer intermediates formed during the oxidative polymerization of DHNs supported this evidence by providing unprecedented insight into the generation and fate of the intermediate naphthoxyl radicals, which emerged as the main factor governing the antioxidant activity of DHNs.

Mining for encrypted peptide antibiotics in the human proteome.

The emergence of drug-resistant bacteria calls for the discovery of new antibiotics. Yet, for decades, traditional discovery strategies have not yielded new classes of antimicrobial. Here, by mining the human proteome via an algorithm that relies on the sequence length, net charge, average hydrophobicity and other physicochemical properties of antimicrobial peptides, we report the identification of 2,603 encrypted peptide antibiotics that are encoded in proteins with biological function unrelated to the immune system. We show that the encrypted peptides kill pathogenic bacteria by targeting their membrane, modulate gut and skin commensals, do not readily select for bacterial resistance, and possess anti-infective activity in skin abscess and thigh infection mouse models. We also show, in vitro and in the two mouse models of infection, that encrypted antibiotic peptides from the same biogeographical area display synergistic antimicrobial activity. Our algorithmic strategy allows for the rapid mining of proteomic data and opens up new routes for the discovery of candidate antibiotics.

Non-covalent small molecule partnership for redox-active films: Beyond polydopamine technology.

HYPOTHESIS: The possibility to use hexamethylenediamine (HMDA) to impart film forming ability to natural polymers including eumelanins and plant polyphenols endowed with biological activity and functional properties has been recently explored with the aim to broaden the potential of polydopamine (PDA)-based films overcoming their inherent limitations. 5,6-dihydroxyindole-2-carboxylic acid, its methyl ester (MeDHICA) and eumelanins thereof were shown to exhibit potent reducing activity. EXPERIMENTS: MeDHICA and HMDA were reacted in aqueous buffer, pH 9.0 in the presence of different substrates to assess the film forming ability. The effect of different reaction parameters (pH, diamine chain length) on film formation was investigated. Voltammetric and AFM /SEM methods were applied for analysis of the film redox activity and morphology. HPLC, MALDI-MS and 1HNMR were used for chemical characterization. The film reducing activity was evaluated in comparison with PDA by chemical assays and using UV stressed human immortalized keratinocytes (HaCat) cells model. FINDINGS: Regular and homogeneous yellowish films were obtained with moderately hydrophobic properties. Film deposition was optimal at pH 9, and specifically induced by HMDA. The film consisted of HMDA and monomeric MeDHICA accompanied by dimers/small oligomers, but no detectable MeDHICA/HMDA covalent conjugation products. Spontaneous assembly of self-organized networks held together mainly by electrostatic interactions of MeDHICA in the anion form and HMDA as the dication is proposed as film deposition mechanism. The film displayed potent reducing properties and exerted significant protective effects from oxidative stress on HaCaT.

π-Electron manipulation of the 5,6-dihydroxyindole/quinone system by 3-alkynylation: mild acid-mediated entry to (cross)-conjugated scaffolds and paradigms for medium-tunable chromophores.

5,6-Dihydroxyindole-based systems engender increasing interest for the design and implementation of new functional aromatic scaffolds and eumelanin-like materials with tailored absorption and electronic properties. However, studies aimed at elucidating the influence of external π-conjugating groups on the redox properties and acid-induced reactivity of these highly oxidizable indolic platforms are lacking. We report herein the synthesis (as acetyl derivatives) and chemical/quantum chemical characterization of the first π-extended 5,6-dihydroxyindole derivatives, 3-ethynyl-5,6-dihydroxyindole (1) and 3,3'-(1,2-ethynediyl)bis-5,6-dihydroxyindole (2), in order to understand whether and how ß extension of the enamine-like pyrrole sector affects the absorption properties, redox behavior, and protonation equilibria at both the o-diphenol and quinone levels. Oxidation of 1 and 2 proceeded smoothly to generate dark insoluble materials with eumelanin-like UV properties. On exposure to phosphate buffer at pH 3, 1 was rapidly converted to 3-acetyl-5,6-dihydroxyindole (5) and, in the presence of 5,6-dihydroxyindole, to the cross-conjugated 3,3'-ethenylidenebis-5,6-dihydroxyindole (6). DFT calculations on 1 and 2 and their quinones in their pristine states and after protonation provided a mechanistic frame to rationalize the unusual acid-mediated chemistry of 1 and disclosed 2-quinone as the prototype of a novel class of medium-dependent chromophores. The ethynyl(ene) structural motif is thus proposed as the key to new tunable π-electron extended 5,6-dihydroxyindole/5,6-indolequinone paradigms for the rational design of alkyne-containing hybrid eumelanin-type polymers.

Oxidation mechanism of diethyl ether: a complex process for a simple molecule.

A large number of organic compounds, such as ethers, spontaneously form unstable peroxides through a self-propagating process of autoxidation (peroxidation). Although the hazards of organic peroxides are well known, the oxidation mechanisms of peroxidizable compounds like ethers reported in the literature are vague and often based on old experiments, carried out in very different conditions (e.g. atmospheric, combustion). With the aim to (partially) fill the lack of information, in this paper we present an extensive Density Functional Theory (DFT) study of autoxidation reaction of diethyl ether (DEE), a chemical that is largely used as solvent in laboratories, and which is considered to be responsible for various accidents. The aim of the work is to investigate the most probable reaction paths involved in the autoxidation process and to identify all potential hazardous intermediates, such as peroxides. Beyond the determination of a complex oxidation mechanism for such a simple molecule, our results suggest that the two main reaction channels open in solution are the direct decomposition (ß-scission) of DEE radical issued of the initiation step and the isomerization of the peroxy radical formed upon oxygen attack (DEEOO˙). A simple kinetic evaluation of these two competing reaction channels hints that radical isomerization may play an unexpectedly important role in the global DEE oxidation process. Finally industrial hazards could be related to the hydroperoxide formation and accumulation during the chain propagation step. The resulting information may contribute to the understanding of the accidental risks associated with the use of diethyl ether.

Extension of the AMBER force-field for the study of large nitroxides in condensed phases: an ab initio parameterization.

The popular AMBER force-field has been extended to provide an accurate description of large and flexible nitroxide free-radicals in condensed phases. New atom types have been included, and relevant parameters have been fitted based on geometries, vibrational frequencies and potential energy surfaces computed at the DFT level for several different classes of nitroxides, both in vacuo and in different solvents. The resulting computational tool is capable of providing reliable structures, vibrational frequencies, relative energies and spectroscopic observables for large and flexible nitroxide systems, including those typically used as spin labels. The modified force field has been employed in the context of an integrated approach, based on classical molecular dynamics and discrete-continuum solvent models, for the investigation of environmental and short-time dynamic effects on the hyperfine and gyromagnetic tensors of PROXYL, TEMPO and INDCO spin probes. The computed magnetic parameters are in very good agreement with the available experimental values, and the procedure allows for an unbiased evaluation of the role of different effects in tuning the overall EPR observables.

Pulse ENDOR and density functional theory on the peridinin triplet state involved in the photo-protective mechanism in the peridinin-chlorophyll a-protein from Amphidinium carterae.

The photoexcited triplet state of the carotenoid peridinin in the Peridinin-chlorophyll a-protein of the dinoflagellate Amphidinium carterae has been investigated by pulse EPR and pulse ENDOR spectroscopies at variable temperatures. This is the first time that the ENDOR spectra of a carotenoid triplet in a naturally occurring light-harvesting complex, populated by energy transfer from the chlorophyll a triplet state, have been reported. From the electron spin echo experiments we have obtained the information on the electron spin polarization dynamics and from Mims ENDOR experiments we have derived the triplet state hyperfine couplings of the alpha- and beta-protons of the peridinin conjugated chain. Assignments of beta-protons belonging to two different methyl groups, with aiso=7.0 MHz and aiso=10.6 MHz respectively, have been made by comparison with the values predicted from density functional theory. Calculations provide a complete picture of the triplet spin density on the peridinin molecule, showing that the triplet spins are delocalized over the whole pi-conjugated system with an alternate pattern, which is lost in the central region of the polyene chain. The ENDOR investigation strongly supports the hypothesis of localization of the triplet state on one peridinin in each subcluster of the PCP complex, as proposed in [Di Valentin et al. Biochim. Biophys. Acta 1777 (2008) 186-195]. High spin density has been found specifically at the carbon atom at position 12 (see Fig. 1B), which for the peridinin involved in the photo-protective mechanism is in close contact with the water ligand to the chlorophyll a pigment. We suggest that this ligated water molecule, placed at the interface between the chlorophyll-peridinin pair, is functioning as a bridge in the triplet-triplet energy transfer between the two pigments.